The mechanism governing differentiation of intestinal epithelial cells is unknown. Due to the extremely complex nature of the mammalian gastrointestinal tract, homogeneous cultured intestinal epithelial cell lines may be more suitable for the initial study of cellular and molecular events that control epithelial differentiation. The human carcinoma cell line HT29 and its clonal derivatives have been shown to exhibit biochemical and morphological characteristics of enterocyte differentiation in vitro and are therefore excellent models for the study of intestinal differentiation. The long-term goal of the proposed project is to define the molecular mechanisms controlling intestinal differentiation in the culture model system of human colon carcinoma cell lines HT29-18 and HT29- 18-C. The major objectives are: 1) to isolate and characterize differentially expressed genes in differentiating HT29-18 cells, and 2) to examine the mechanism regulating transcription of these genes in differentiating HT29-18 cells. Using the molecular technique of subtraction cDNA library screening, we have already isolated a full-length cDNA clone (A4) representing a novel gene whose expression is differentially regulated in HT29-18 and HT29-18-C cells. The A4 cDNA represents a messenger RNA of 1.0 kb in size whose abundance is dramatically induced as HT29-18 cells differentiate. Amino acid sequences deduced from A4 nucleotide sequences have a limited homology to a portion of the cytoskeletal protein, ankyrin. The expression of A4 is enriched in colonic mucosa and is transcriptionally activated during HT29 differentiation. We have also shown that the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR) is also transcriptionally activated during HT29 differentiation. We now plan to perform the following experiments: 1) to isolate by subtraction cloning additional cDNA clones that are transcriptionally activated, 2) to characterize the newly identified differentiation-dependent genes, and 3) to investigate the mechanisms regulating transcription of differentially expressed genes including A4, CFTR and genes obtained in (1). By performing these experiments, we hope to identify a concerted mechanism regulating transcription of a selected group of genes in a culture model of intestinal epithelial differentiation.